Field of the Intention
The present invention relates to a method for preparing peptide fragments by site-selectively proteolyzing a protein, such as an antibody, using a protease, and a kit for preparing peptide fragments to be used therein. Further, the present invention relates to a method for analyzing peptide fragments, prepared by the method, by mass spectrometry or the like to detect or quantitate a protein.
Discussion of the Background
A method has been developed in which a protein to be analyzed is subjected to site-selective proteolysis to reduce the number (of types) of peptide fragments in a sample to improve the accuracy of analysis and simplify the process of analysis. For example, WO 2008/079914 proposes a method in which an antibody is subjected to pepsin digestion to produce an F(ab′)2 fragment, and then the F(ab′)2 fragment is further digested with a protease such as trypsin to produce peptide fragments containing the complementarity-determining region (CDR) of the antibody, and the peptide fragments containing CDR are detected and quantitated by mass spectrometry. JP 2011-130749 A reports that combined use of pepsin or papain and a specific ion improves the efficiency of proteolysis with such a protease.
Fei Xu et. al., Anal. Chem., 2010, 82, 10045-10051 report an example in which the efficiency of trypsin digestion of albumin is increased by allowing an albumin solution to pass through a nylon porous membrane having trypsin immobilized in pores thereof. Qianhao Min et. al., Chem. Commun., 2010, 46(33), 6144-6146 report that a protein having a small molecular weight can be selectively subjected to trypsin digestion by using mesoporous silica having trypsin immobilized in pores thereof.
Hereinafter, the structure of an antibody will be described. All antibodies have two heavy chains (H chains) and two light chains (L chains). One light chain and one heavy chain are linked through a disulfide (S—S) bond to form a heterodimer, and the two heterodimers are further linked through two disulfide bonds to form a “Y”-shaped heterotetramer (see FIG. 2). An antibody has one Fc (Fragment, crystallizable) domain comprising heavy chains and two Fab (Fragment, antigen binding) domains comprising a heavy chain and a light chain, and the Fc domain and the Fab domains are linked through a hinge region.
The Fc domain of an antibody mainly has the function of initiating a reaction after the antibody binds to an antigen (effector function), and most of antibodies derived from the same species have a common amino acid sequence in the Fc domain. On the other hand, the end (on the N-terminal side) of the Fab domain has the function of binding to an antigen. The N-terminal part of the Fab domain diversely changes in its amino acid sequence so as to be able to bind to various antigens. This region is called variable region (V region), and the variable region of the light chain and the variable region of the heavy chain are called VL region and VH region, respectively. The Fab and Fc domains other than the V region are called constant region (C region) that varies little in amino acid sequence. The constant region of the light chain is called CL region, and the constant region of the heavy chain is called CH region. The CH region is further divided into three regions, CH1 region, CH2 region, and CH3 region. The Fab domain of the heavy chain comprises the VH region and the CH1 region, and the Fc domain of the heavy chain comprises CH2 and CH3. The hinge region is located between CH1 and CH2.
The specificity (i.e., specific bindability to an antigen) of an antibody is determined by the combination of amino acid sequences of the V region. The light chain and the heavy chain each have three complementarity-determining regions (CDRs) in the V region of the Fab domain. CDR is also called hypervariable region, and varies in amino acid sequence depending on the type of antibody. There are 3 CDRs on each of the heavy and light chains of an antibody (6 types of CDRs in total), which creates diversity that allows the antibody to bind to various antigens. In other words, CDRs are regions characterizing an antibody, and therefore an antibody can be identified by identifying the amino acid sequences of CDRs thereof.
As described above, the Fab domains and Fc domain of an antibody are linked through a hinge region. Papain which is a kind of protease proteolyzes the hinge region, and therefore two Fab domains and one Fc domain are produced by papain digestion of an antibody. Further, pepsin which is a kind of protease proteolyzes one of the two disulfide bonds, i.e., the Fc domain-side (C-terminal side) disulfide bond of the hinge region, and therefore an F(ab′)2 domain having two Fab domains linked together and many Fc domain fragments are produced by pepsin digestion.